Benzimidazole-Based FabI Inhibitors: A Promising Novel Scaffold for Anti-staphylococcal Drug Development.

The enoyl-ACP reductase (FabI) enzyme is a well validated target for anti-staphylococcal drug discovery and development. With the goal of finding alternate therapeutics for drug-resistant strains of Staphylococcus aureus, such as methicillin-resistant S. aureus (MRSA), our previously published series of benzimidazole-based inhibitors of the FabI enzyme from Francisella tularensis (FtFabI) have been evaluated against FabI from S. aureus (SaFabI). We report here the preliminary structure-activity relationship of this series and the prioritization of compounds toward lead optimization. Mutational studies have identified key residues that contribute toward stabilizing the inhibitors in the active site of FabI. Mutations that do not significantly impact enzyme function but destabilize inhibitor binding are more likely to occur in nature as organisms evolve to evade the action of antibiotics leading to resistance. Identifying these residues provides guidance for minimizing susceptibility to resistance. Additionally, we have identified compounds that elicit antibacterial activity through off-target effects and observe that close analogs can display differing modes of action (on-target vs off-target) and need to be individually evaluated early on to prioritize compounds for lead optimization. Overall, our data suggest that the benzimidazole scaffold is a promising scaffold for anti-staphylococcal drug development.

Structural and biological evaluation of a novel series of benzimidazole inhibitors of Francisella tularensis enoyl-ACP reductase (FabI).

Francisella tularensis, the causative agent of tularemia, presents a significant biological threat and is a Category A priority pathogen due to its potential for weaponization. The bacterial FASII pathway is a viable target for the development of novel antibacterial agents treating Gram-negative infections. Here we report the advancement of a promising series of benzimidazole FabI (enoyl-ACP reductase) inhibitors to a second-generation using a systematic, structure-guided lead optimization strategy, and the determination of several co-crystal structures that confirm the binding mode of designed inhibitors. These compounds display an improved low nanomolar enzymatic activity as well as promising low microgram/mL antibacterial activity against both F. tularensis and Staphylococcus aureus and its methicillin-resistant strain (MRSA). The improvements in activity accompanying structural modifications lead to a better understanding of the relationship between the chemical structure and biological activity that encompasses both enzymatic and whole-cell activity.

Identification of novel drug scaffolds for inhibition of SARS-CoV 3-Chymotrypsin-like protease using virtual and high-throughput screenings.

We have used a combination of virtual screening (VS) and high-throughput screening (HTS) techniques to identify novel, non-peptidic small molecule inhibitors against human SARS-CoV 3CLpro. A structure-based VS approach integrating docking and pharmacophore based methods was employed to computationally screen 621,000 compounds from the ZINC library. The screening protocol was validated using known 3CLpro inhibitors and was optimized for speed, improved selectivity, and for accommodating receptor flexibility. Subsequently, a fluorescence-based enzymatic HTS assay was developed and optimized to experimentally screen approximately 41,000 compounds from four structurally diverse libraries chosen mainly based on the VS results. False positives from initial HTS hits were eliminated by a secondary orthogonal binding analysis using surface plasmon resonance (SPR). The campaign identified a reversible small molecule inhibitor exhibiting mixed-type inhibition with a K(i) value of 11.1 µM. Together, these results validate our protocols as suitable approaches to screen virtual and chemical libraries, and the newly identified compound reported in our study represents a promising structural scaffold to pursue for further SARS-CoV 3CLpro inhibitor development.

High-throughput screening (HTS) and hit validation to identify small molecule inhibitors with activity against NS3/4A proteases from multiple hepatitis C virus genotypes.

Development of drug-resistant mutations has been a major problem with all currently developed Hepatitis C Virus (HCV) NS3/4A inhibitors, including the two FDA approved drugs, significantly reducing the efficacy of these inhibitors. The high incidence of drug-resistance mutations and the limited utility of these inhibitors against only genotype 1 highlight the need for novel, broad-spectrum HCV therapies. Here we used high-throughput screening (HTS) to identify low molecular weight inhibitors against NS3/4A from multiple genotypes. A total of 40,967 compounds from four structurally diverse molecular libraries were screened by HTS using fluorescence-based enzymatic assays, followed by an orthogonal binding analysis using surface plasmon resonance (SPR) to eliminate false positives. A novel small molecule compound was identified with an IC50 value of 2.2 µM against the NS3/4A from genotype 1b. Mode of inhibition analysis subsequently confirmed this compound to be a competitive inhibitor with respect to the substrate, indicating direct binding to the protease active site, rather than to the allosteric binding pocket that was discovered to be the binding site of a few recently discovered small molecule inhibitors. This newly discovered inhibitor also showed promising inhibitory activity against the NS3/4As from three other HCV genotypes, as well as five common drug-resistant mutants of genotype 1b NS3/4A. The inhibitor was selective for NS3 from multiple HCV genotypes over two human serine proteases, and a whole cell lysate assay confirmed inhibitory activity in the cellular environment. This compound provides a lead for further development of potentially broader spectrum inhibitors.

A colorimetric assay optimization for high-throughput screening of dihydroorotase by detecting ureido groups.

Dihydroorotase (DHOase) is the third enzyme in the de novo pyrimidine biosynthesis pathway and is a potential new antibacterial drug target. No target-based high-throughput screening (HTS) assay for this enzyme has been reported to date. Here, we optimized two colorimetric-based enzymatic assays that detect the ureido moiety of the DHOase substrate, carbamyl-aspartate (Ca-asp). Each assay was developed in a 40-µl assay volume using 384-well plates with a different color mix, diacetylmonoxime (DAMO)-thiosemicarbazide (TSC) or DAMO-antipyrine. The sensitivity and color interference of both color mixes were compared in the presence of common HTS buffer additives, including dimethyl sulfoxide, reducing agents, detergents, and bovine serum albumin. DAMO-TSC (Z'-factors 0.7-0.8) was determined to be superior to DAMO-antipyrine (Z'-factors 0.5-0.6) with significantly less variability within replicates. An HTS pilot screening with 29,552 compounds from four structurally diverse libraries confirmed the quality of our newly optimized colorimetric assay with DAMO-TSC. This robust method has no heating requirement, which was the main obstacle to applying previous assays to HTS. More important, this well-optimized HTS assay for DHOase, the first of its kind, should make it possible to screen large-scale compound libraries to develop new inhibitors against any enzymes that produce ureido functional groups.

Synergistic inhibitor binding to the papain-like protease of human SARS coronavirus: mechanistic and inhibitor design implications.

We previously developed two potent chemical classes that inhibit the essential papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus. In this study, we applied a novel approach to identify small fragments that act synergistically with these inhibitors. A fragment library was screened in combination with four previously developed lead inhibitors by fluorescence-based enzymatic assays. Several fragment compounds synergistically enhanced the inhibitory activity of the lead inhibitors by approximately an order of magnitude. Surface plasmon resonance measurements showed that three fragments bind specifically to the PLpro enzyme. Mode of inhibition, computational solvent mapping, and molecular docking studies suggest that these fragments bind adjacent to the binding site of the lead inhibitors and further stabilize the inhibitor-bound state. We propose potential next-generation compounds based on a computational fragment-merging approach. This approach provides an alternative strategy for lead optimization for cases in which direct co-crystallization is difficult.

High-level expression, purification, and characterization of Staphylococcus aureus dihydroorotase (PyrC) as a cleavable His-SUMO fusion.

Staphylococcus aureus is a pathogenic bacterium that causes a variety of mild to lethal human diseases. The rapid spread of multidrug-resistant strains makes the discovery of new antimicrobial agents critical. Dihydroorotase (PyrC), the third enzyme in the bacterial pyrimidine biosynthesis pathway, is structurally and mechanistically distinct from its mammalian counterpart. It has been confirmed to be essential in S. aureus making it an attractive antibacterial drug target. No protocol to express and purify S. aureus PyrC (SaPyrC) has been reported. To obtain the SaPyrC enzyme and overcome anticipated solubility problems, the SaPyrC gene was cloned into the pET-SUMO vector. The N-terminal His-SUMO fused SaPyrC was expressed in Escherichia coli BL21 (DE3) with an HRV 3C protease recognition site inserted between the SUMO tag and SaPyrC to allow for improved cleavage by HRV protease. Purification of cleaved protein using HisTrap affinity and gel filtration columns resulted in native SaPyrC with estimated 95% purity and 40% yield. Both His-SUMO tagged and native SaPyrC form dimers, and enzyme characterization studies have shown that the His-SUMO tag affects enzyme activity slightly. Forward and reverse kinetic rate constants for both tagged and native SaPyrC were determined, and pH profiling studies revealed the optimal pH values for forward and reverse reactions.

Identification of non-macrocyclic small molecule inhibitors against the NS3/4A serine protease of hepatitis C virus through in silico screening.

Drug discovery and design for inhibition of the Hepatitis C Virus (HCV) NS3/4A serine protease is a major challenge. The broad, shallow, and generally featureless nature of the active site makes it a difficult target for "hit" selection especially using standard docking programs. There are several macrocyclic NS3/4A protease inhibitors that have been approved or are in clinical trials to treat chronic HCV (alone or as combination therapy), but most of the current therapies for HCV infection have untoward side effects, indicating a continuing medical need for the discovery of novel therapeutics with improved efficacy. In this study, we designed and implemented a two-tiered and progressive docking regime that successfully identified five non-macrocyclic small molecules that show inhibitory activity in the low micromolar range. Of these, four compounds show varying inhibition against HCV subgenotypes 1b, 1a, 2a, and 4d. The top inhibitor (3) has an IC(50) value of 15 µM against both subgenotypes 1b and 2a of the NS3/4A protease enzyme. Another inhibitor, 1, inhibits all four subgenotypes with moderate activity, showing highest activity for genotype 2a (24 µM). The five inhibitors presented in this study could be valuable candidates for future hit to lead optimization. Additionally, enzyme-inhibitor interaction models presented herein provide key information regarding structural differences between the active sites of the NS3/4A protease of the HCV subgenotype 1a and 1b that might explain the variable inhibitory activity between subgenotypes of the small molecule inhibitors identified here.

Reducing agents affect inhibitory activities of compounds: results from multiple drug targets.

High-throughput screening (HTS) of large compound libraries has become a commonly used method for the identification of drug leads, and nonphysiological reducing agents have been widely used for HTS. However, a comparison of the difference in the HTS results based on the choice of reducing agent used and potency comparisons of selected inhibitors has not been done with the physiological reducing agent reduced glutathione (GSH). Here, we compared the effects of three reducing agents-dithiothreitol (DTT), ß-mercaptoethanol (ß-MCE), and tris(2-carboxyethyl)phosphine (TCEP)-as well as GSH against three drug target proteins. Approximately 100,000 compounds were computationally screened for each target protein, and experimental testing of high-scoring compounds (~560 compounds) with the four reducing agents surprisingly produced many nonoverlapping hits. More importantly, we found that various reducing agents altered inhibitor potency (IC(50)) from approximately 10 µM with one reducing agent to complete loss (IC(50)>200 µM) of inhibitory activity with another reducing agent. Therefore, the choice of reducing agent in an HTS is critical because this may lead to the pursuit of falsely identified active compounds or failure to identify the true active compounds. We demonstrate the feasibility of using GSH for in vitro HTS assays with these three target enzymes.